Engineered P450 BM3 and cpADH5 coupled cascade reaction for β-oxo fatty acid methyl ester production in whole cells,Enzyme and Microbial Technology

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Engineered P450 BM3 and cpADH5 coupled cascade reaction for β-oxo fatty acid methyl ester production in whole cells
Enzyme and Microbial Technology ( IF 3.4 ) Pub Date : 2020-08-01 , DOI: 10.1016/j.enzmictec.2020.109555
Yunus Ensari ₁ , Gustavo de Almeida Santos ₂ , Anna Joëlle Ruff ₂ , Ulrich Schwaneberg ₃

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Hydroxy- or ketone- functionalized fatty acid methyl esters (FAMEs) are important compounds for production of pharmaceuticals, vitamins, cosmetics or dietary supplements. Biocatalysis through enzymatic cascades has drawn attention to the efficient, sustainable, and greener synthetic processes. Furthermore, whole cell catalysts offer important advantages such as cofactor regeneration by cell metabolism, omission of protein purification steps and increased enzyme stability. Here, we report the first whole cell catalysis employing an engineered P450 BM3 variant and cpADH5 coupled cascade reaction for the biosynthesis of hydroxy- and keto-FAMEs. Firstly, P450 BM3 was engineered through the KnowVolution approach yielding P450 BM3 variant YE_M1_2, (R47S/Y51W/T235S/N239R/I401 M) which exhibited boosted performance toward methyl hexanoate. The initial oxidation rate of YE_M1_2 toward methyl hexanoate was determined to be 23-fold higher than the wild type enzyme and a 1.5-fold increase in methyl 3-hydroxyhexanoate production was obtained (YE_M1_2; 2.75 mM and WT; 1.8 mM). Subsequently, the whole cell catalyst for the synthesis of methyl 3-hydroxyhexanoate and methyl 3-oxohexanoate was constructed by combining the engineered P450 BM3 and cpADH5 variants in an artificial operon. A 2.06 mM total product formation was achieved by the whole cell catalyst including co-expressed channel protein, FhuA and co-solvent addition. Moreover, the generated whole cell biocatalyst also accepted methyl valerate, methyl heptanoate as well as methyl octanoate as substrates and yielded ω-1 ketones as the main product.

中文翻译：

工程化 P450 BM3 和 cpADH5 偶联级联反应，用于在全细胞中生产 β-氧代脂肪酸甲酯

羟基或酮官能化脂肪酸甲酯 (FAME) 是用于生产药物、维生素、化妆品或膳食补充剂的重要化合物。通过酶促级联的生物催化已引起人们对高效、可持续和更环保的合成过程的关注。此外，全细胞催化剂具有重要的优势，例如通过细胞代谢进行辅因子再生、省略蛋白质纯化步骤和增加酶稳定性。在这里，我们报告了第一个使用工程化 P450 BM3 变体和 cpADH5 偶联级联反应进行羟基和酮基 FAME 生物合成的全细胞催化。首先，P450 BM3 是通过 KnowVolution 方法设计的，产生了 P450 BM3 变体 YE_M1_2（R47S/Y51W/T235S/N239R/I401 M），其表现出对己酸甲酯的增强性能。YE_M1_2 对己酸甲酯的初始氧化速率被确定为比野生型酶高 23 倍，并且 3-羟基己酸甲酯的产量增加了 1.5 倍（YE_M1_2；2.75 mM 和 WT；1.8 mM）。随后，通过在人工操纵子中结合工程化的 P450 BM3 和 cpADH5 变体，构建了用于合成 3-羟基己酸甲酯和 3-氧代己酸甲酯的全细胞催化剂。通过包括共表达的通道蛋白、FhuA 和共溶剂添加在内的全细胞催化剂实现了 2.06 mM 的总产物形成。此外，生成的全细胞生物催化剂还接受戊酸甲酯、庚酸甲酯和辛酸甲酯作为底物，并产生 ω-1 酮作为主要产物。

更新日期：2020-08-01

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